Spinal cord injury (SCI) is a devastating traumatic injury for which there currently is no cure. SCI triggers a robust inflammatory response that is dominated by activated CNS macrophages. Data suggests that modulating macrophage activity after SCI can limit delayed neurodegeneration and promote functional recovery. Studies in this proposal will examine how the lesion microenvironment drives macrophage inflammatory function. HIF1 is a ubiquitous transcription factor regulating the cellular response to hypoxia. Within macrophages however, it has also been shown to be crucial for both resting metabolism and inflammatory function. Studies in Aim 1 will provide novel data on the regulation of HIF1a and its downstream effectors in cultured macrophages in response to hypoxic and inflammatory stimuli, and what role this regulation plays in macrophage-mediated neurite outgrowth and neurotoxicity. Use of knockout mice with macrophage-specific deletion of HIF1a will allow us to tease HIF1a-dependent regulation from other inflammatory signaling cascades. Production of cytokines and other inflammatory mediators will be assayed via real-time RT-PCR and Western blotting. Macrophage-mediated neuroregeneration and neurotoxicity we be assessed in vitro using established neurite outgrowth and neuron death assays. In parallel, studiles in Aim 2 will use a clinically relevant mouse model of contusive SCI to compare lesion progression and functional recovery between mice expressing WT or KO microglia/macrophages. HIF1a's role in the macrophage response to SCI will be assessed through stereological measurements of lesion volume, myelin sparing, and macrophage accumulation. Immunohistochemistry and laser capture microscopy will be used to define and quantify cell-specific expression of HIF1a, its major downstream targets, and proinflammatory cytokines in injured and uninjured spinal cord sections. Permeability studies will be used to determine if macrophage-expressed downstream genes of HIF1a effect affect blood-spinal cord barrier integrity. Locomotor behavior tests will be performed to detect differences in functional recovery between the groups. Understanding the complex and multi-faceted regulation of HIF1a in the unique microenvironments present in the SCI lesion will give researchers and clinicians insight into the roles of inflammation post-SCI. We believe that therapies designed to attenuate or manipulate subacute and chronic disease processes such as ischemia-reperfusion injury and inflammation will improve functional outcome in SCI patients, thus improving their quality of life and reducing the immense social and economic burden associated with this disorder.
|Donnelly, Dustin J; Longbrake, Erin E; Shawler, Todd M et al. (2011) Deficient CX3CR1 signaling promotes recovery after mouse spinal cord injury by limiting the recruitment and activation of Ly6Clo/iNOS+ macrophages. J Neurosci 31:9910-22|